Protein folding is uniquely characterized as a dynamic process by the tremendous conformational heterogeneity of the unfolded state. This conformational heterogeneity implies the possibility of significant kinetic heterogeneity in the folding dynamics, yet many folding reactions appear to proceed homogeneously, i.e., from unfolded conformations that are in equilibrium with each other and a transition state. At what point in time on the way to the folded state the kinetic heterogeneity of the unfolded state is lost to conformational equilibration has been an open question. We present evidence in this regard obtained from nanosecond far-UV optical rotatory dispersion spectroscopy of the fastest folding process observed in photoreduced cytochrome c, a secondary structure formation process proceeding on a submicrosecond time scale at high denaturant concentration (observed as a "burst" phase in millisecond stopped-flow circular dichroism studies). The kinetics of this fast folding process imply that it proceeds from a conformational ensemble that is not in equilibrium with the bulk of protein conformers during the time required to completely reduce the sample, similar to100 mus. We thus determine a lower limit on the time required for conformational equilibration of similar to10(-4) s, in agreement with the previous estimate from time-resolved magnetic circular dichroism measurements on photolyzed cytochrome c-CO (Goldbeck, R. A.; Thomas, Y. G.; Chen, E.; Esquerra, R. M.; Kliger, D. S. Proc. Natl. Acad. Sci. 1999, 96, 2782-2787). Combined with an upper limit from stopped-flow measurements (Lyubovitsky, J. G.; Gray, H. B.; Winkler, J. R. J. Am. Chem. Soc. 2002, 124, 5481-5495), this allows us to bracket the conformational diffusion time, i.e., the time interval over which the kinetic heterogeneity of this protein's unfolded state is lost through conformational equilibration, within the range similar to10(-4)-10(-3) s. This estimate of the conformational diffusion time implies that the earliest folding events, helix formation and possibly the collapse of extended conformations, proceed under an energy landscape regime wherein conformational diffusion is slow, whereas the final (milliseconds) folding phase proceeds along a classical kinetic pathway.